Collaborative Research: Iron limitation, carbon metabolism and siderophore production in marine bacteria - a systems biology approach

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。异养细菌的铁限制具有重要的生物地球化学意义，包括降低同化效率和减少二氧化碳进入生物质。海洋浮游细菌也通过产生铁载体（对铁具有高亲和力的配体）对海水中铁的形成产生很大影响。海水中超过99.9%的溶解铁与有机配体紧密结合，其中可能包括铁载体，因为它们具有与原位螯合剂相似的官能团和铁的条件稳定常数。铁限制和铁载体合成是联系在一起的，因为这些配体是在细胞内铁浓度低的情况下特异性产生的。这两个过程在海洋细菌中都没有被完全理解。例如，当铁加入到HNLC区域时，海水中铁结合配体的产生出乎意料地增加，而不是减少。一个可能的解释是碳的作用，它使铁限制和铁载体合成的研究复杂化。低铁浓度对浮游细菌原位生长效率或生产力的影响程度尚未得到很好的量化，因为在浮游植物的铁限制解除后，初级生产会迅速受到刺激。由此产生的可用碳的增加使得很难区分海洋细菌的直接和间接铁（即碳）限制。碳源和可利用性也可能在调节铁载体的产生中起次要但重要的作用，而铁载体的产生可以通过添加葡萄糖来刺激或抑制。本项目的目的是模拟γ -变形菌中控制铁获取和碳代谢的相互作用基因调控网络，特别是费氏弧菌。铁和碳调控途径在一个由全局转录调控因子（Fur和CRP）和小RNA RyhB介导的复杂关系网络中紧密相连。为了构建基因调控网络模型，pi将使用综合系统生物学方法，将计算、生物信息学研究和实验室实验相结合。通过使用qRT-PCR和全球转录组分析定量基因表达，以及确定铁配额和铁摄取速率，将验证基因表达、铁载体产生和铁通量随环境条件变化的预测。知识价值：本研究将通过阐明铁与碳限制、碳源和铁载体生产之间的相互作用，有助于对上层海洋铁形态的一般理解。基因调控网络模型将能够识别对铁载体产生至关重要的环境变量，并评估铁限制的潜在生物标志物。该项目将有助于发现涉及铁代谢的新基因和控制机制，并揭示利用相同或类似的相互作用和转录因子的其他过程，如毒力和发光。最后，这种方法还提供了一个框架，用于综合遗传水平上的信息，并利用它来预测诸如铁载体生产等具有生态重要性的过程。更广泛的影响：该项目将通过指导青年科学家、支持多个教育层次的推广项目和课程开发，加强科学研究和教育的基础设施。要求支持研究生参与拟议的研究，所有三个pi都将招募本科生进行夏季实习，通常针对代表性不足的群体。教师研究经验补品要求允许两位高中科学教育工作者参与我们在Skidaway海洋学研究所的研究，并设计一个实验室练习，以说明微生物如何对营养限制作出反应。其目的是提供一种研究经验，以加强地方和国家各级教育工作者之间的合作。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Iron limitation of heterotrophic bacteria has substantial biogeochemical implications, including lower assimilation efficiencies and reduced incorporation of CO2 into biomass. Marine bacterioplankton also have a large impact on iron speciation in seawater through their production of siderophores, ligands with a high affinity for iron. Over 99.9% of the dissolved iron in seawater is strongly bound to organic ligands, which are likely to include siderophores since they have functional groups and conditional stability constants for iron that are similar to chelators detected in situ. Iron limitation and siderophore synthesis are linked since these ligands are produced specifically in response to low intracellular iron concentrations. Neither process is completely understood in marine bacteria. For instance, the production of iron binding ligands in seawater unexpectedly increases, not decreases when iron is added to HNLC regions. One possible explanation is the role of carbon, which complicates studies of both iron limitation and siderophore synthesis. The extent to which low iron concentrations reduce bacterioplankton growth efficiencies or productivity in situ is not well-quantified because of the rapid stimulation of primary production that occurs after iron limitation of phytoplankton is relieved. The resulting increase in available carbon makes it is difficult to distinguish between direct and indirect iron (i.e. carbon) limitation of marine bacteria. Carbon source and availability may also play a secondary but important role in regulating siderophore production, which can be either stimulated or repressed by the addition of glucose. The objective of this project is to model the interacting gene regulatory networks that control iron acquisition and carbon metabolism in gamma -proteobacteria, specifically Vibrio fischeri. Iron and carbon regulatory pathways are tightly linked in a complex web of relationships mediated by global transcriptional regulators (Fur and CRP) and the small RNA RyhB. In order to construct a gene regulatory network model, the PIs will use an integrated systems biology approach that combines computational, bioinformatics based research and laboratory experimentation. Predictions of gene expression, siderophore production and the flux of iron with changing environmental conditions will be validated by quantifying gene expression using qRT-PCR and global transcriptome analyses, as well as determining iron quotas and iron uptake rates. Intellectual Merit: This study will contribute to the general understanding of iron speciation in the upper ocean by elucidating the interactions between iron and carbon limitation, carbon source and siderophore production. The gene regulatory network model will be capable of identifying environmental variables critical to siderophore production and evaluating potential biomarkers for iron limitation. This project will facilitate the discovery of new genes and control mechanisms involved in iron metabolism and shed light on other processes such as virulence and luminescence, which utilize the same or similar interactions and transcription factors. Finally, this approach also provides a framework for synthesizing information on the genetic level and using it to make predictions about processes such as siderophore production that are ecologically important. Broader Impacts: This project will enhance infrastructure for scientific research and education by mentoring young scientists and supporting both outreach programs and course development at several educational levels. Support is requested for graduate students to participate in the proposed research and all three PIs will recruit undergraduates for summer internships, often targeting underrepresented groups. Research Experiences for Teachers supplements are requested to allow two high school science educators to participate in our research at the Skidaway Institute of Oceanography and design a laboratory exercise that will illustrate how microbes respond to nutrient limitation. The aim is to provide a research experience that will strengthen collaborations among educators at both the local and national levels.